Cell culture is the complex process by which cells are grown under controlled conditions, generally outside of their natural environment. In practice cell culture refers to the culturing of cells derived from multi-cellular eukaryotes, especially animal cells. However, there are also cultures of plants, fungi, insects and microbes, including viruses, bacteria and protista.
Cells are grown and maintained at an appropriate temperature and gas mixture (typically, 37° C., 5% CO2 for mammalian cells) in a cell incubator. Culture conditions vary widely for each cell type, and variation of conditions for a particular cell type can result in different phenotypes.
A variety of cell types are grown in vitro and similarly a variety of media are available depending on the particular growth requirements of the cells and the growth conditions.
T cells or T lymphocytes are a type of lymphocyte that play a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T-cell receptor (TCR) on the cell surface. They are called T cells because they mature in the thymus. There are several subsets of T cells, each with a distinct function, including T helper cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and mucosal associated invariant T cells.
Many devices have been developed for culturing cells, which can be divided into three categories: 1) small-scale devices with a culture volume typically between 1 L to 20 L, although culture volumes of less than 1 L, even in the range of 10 mL or 100 mL are not exceptional; 2) medium-scale devices with culture volumes between 20 and 2,000 L; and 3) large-scale bioreactors with operating volumes from 2,000 up to 20,000 L. In general, small-scale devices are limited to a few liters in volume because they rely on surface oxygen transfer to provide aeration for cells. Examples of small-scale devices include multiple well tissue culture plates, spinner flasks, T-flasks, roller bottles, and cell culture bags.
Commercially available gas permeable cell culture devices in the form of bags are currently a standard device format used for cell culture. Cell culture bags have the advantage of being disposable, which reduces preparation and clean up time. Additionally, cell culture bags are pre-sterilizable, inexpensive, easy to use and require minimal space for storage and use.
Gas permeable fluoropolymer cell culture bags are commercially available from OriGen Biomedical Group (OriGen PermaLife™ Bags), Baxter (Lifecell® X-Fold™ related to U.S. Pat. Nos. 4,829,002, 4,937,194, 5,935,847, 6,297,046 B1), Medtronic (Si-Culture™, U.S. Pat. No. 5,686,304), Biovectra (VectraCell™), and American Fluoroseal (VueLife™ Culture Bag System, covered by U.S. Pat. Nos. 4,847,462 and 4,945,203).
One problem of conventional fluoropolymer cell culture bags is that the feed media and cells are mixed within the culture chamber and therefore the amount of media added is limited to the volume of the container. In one example, Matsumiya et al. (U.S. Pat. No. 5,225,346) attempts to correct the problem of media supply by integrating the bag with a medium storage compartment. The culture chamber and medium storage compartment are connected and when fresh medium is needed it is passed from the medium storage compartment to the culture chamber.
Another problem of conventional fluoropolymer cell culture bags is removal of media having diminished nutrient content and increased waste content during cell cultivation which unavailingly removes the desired cells as well.
Therefore, a need exists for the removal and replenishment of feed media without diminishing cell count thereby advantageously concentrating cells over a period of time not controlled by feed media supply limitations.